Lsm 700 confocal

The At4CL1p:GFP (Taylor-Teeples et al., 2015 (link)) and MSL4p:GFP-GUS (gift of Elizabeth Haswell) lines were grown as described above using 1% sucrose. Plants were harvested after 5 days and imaged using a Zeiss LSM 700 Confocal. Images were captured at 20X magnification. In order to capture the length of the whole root at this magnification, multiple images were obtained at exactly the same gain settings and stitched together in Affinity Design into a single image. In order to improve visibility of GFP reporter abundance, brightness was adjusted for the entire stitched images.

Cell proliferation was determined by Ki67 immunofluorescence staining and by live cell counting. Following transfection with the indicated miRNA or siRNA, HLMVEC were seeded (0.3 × 10⁵ cells per well) onto fibronectin-coated (1μg/cm²) Nunc Lab-Tek II chamber slides (Thermo Fisher) for immunostaining. After 24 hours, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min at room temperature. Ki67 was stained with Alexa 488-conjugated mouse-anti human Ki67 antibody (sc-23900 AF488, Santa Cruz) and mounted with ProLong Gold with DAPI (Thermo Fisher). For cell counting, 10–15 images were taken per condition in a blinded fashion with Nikon Eclipse Ti-U inverted microscope with Nikon Elements BR v4.30.02 software. Ki67 positive cells and total number of cells (DAPI positive) were counted using Image J (1.52p) and the data were expressed as percentages of Ki67 positive cells. Representative images for figures were taken with Zeiss LSM700 Confocal with Zen Black software. For growth rate studies, transfected cells were seeded at 1.5 × 10⁴/ well on gelatin-coated 24-well plates. After 24, 48 and 72 hours, cells were stained with Trypan blue stain (ThermoFisher, 0.4%) and counted using a hematocytometer. Each independent experiment was performed in technical duplicate.

For FRAP experiments, embryos were electroporated with GFP-Myosin at stage X and then incubated for 5h until clear cortical GFP-Myosin signal could be visualized. For stage 3, the same embryos where FRAP was performed at stage X were kept incubated overnight for a total incubation time of 14h. The GFP-Myosin signal did not show any significant difference in intensity between both stages and identical photobleaching and imaging conditions were used. A 10x10 pixels cortical region was photobleached using a 488nm laser at 100% (pixel dwell time of 100 μs for 10 iterations). Cells were then imaged every 1.5s for at least 250s using a Zeiss LSM700 confocal and a 40x objective. All quantifications were performed in Fiji. Images were adjusted for xy drift with the plugin: “Linear Stack Alignment with SIFT” using a translational transformation. ROIs within the bleached and within non-bleached areas were then manually selected in order to compensate for acquisition bleach and to normalize the values. The curve fitting of the data was done using a custom made plugin in Matlab (MathWorks). Final plots were done using Prism (GraphPad Software).